How it Works: Fluorescence Mix-and-read Assays for Antibody Discovery

Problem: The therapeutic use of monoclonal antibodies has grown over the last decade and their availability has profoundly modified the treatment of a number of conditions including cancer and autoimmune diseases. The generation of thousands of highly diverse cells secreting antibodies with a range of affinities and concentrations presents a challenge for the rapid selection of those cells producing antibodies of interest. Traditionally, the enzyme-linked immunosorbent assay (ELISA) has been the screening assay of choice. However, with the numerous wash and incubation steps, this process can be tedious and time-consuming. Moreover, this method has limitations when it comes to detecting low abundance proteins, such as cell surface antigens, and there have been concerns that adsorption of antigens onto microtiter plates can alter the protein conformation and lead to failure of identifying antibodies for target epitopes. For these reasons, alternative screening technologies are being sought as the biopharmaceutical industry faces the challenges presented by increasingly complex therapeutic targets.

Solution: Efficient antibody screening requires an assay that is flexible, robust and suitable for automation. TTP LabTech has developed both bead- and cell-based fluorescence methods for the screening of antibodies against soluble and cell surface antigens. These methods combine fluorescence probes with novel laser scanning instrumentation to produce so-called ‘mix-and-read assays’. The basic assay set-up involves the mixing of three components: a sample of cell supernatant containing test antibody; fluorescent tracer to report binding (typically a labelled anti-species antibody); and either cells expressing the antigen of choice or beads coated with the relevant soluble antigen. All assay constituents are incubated together until they have reached equilibrium. Analysis is then performed on Mirrorball™, a new highsensitivity laser scanning microplate cytometer, which reports the amount of fluorescence bound to each bead or cell, thus providing an index of the degree of antibody binding. The unique feature of these mix-and-read protocols is that there is no requirement to remove unbound fluorescent tracer once equilibrium has been reached—the plates are simply analysed after the required time period. These assays thus eliminate the need for wash and incubation steps, resulting in higher throughput, reduced cell loss and lower assay costs due to the smaller volume of reagents used.

Mirrorball has a unique set of features that make this system especially wellsuited to perform mix-and-read assays. The system has high performance lowloss optics which are sensitive enough to enable detection of low abundance proteins. The simultaneous laser scanning functionality results in enhanced multiplexing and allows direct correlation of data across lasers leading to more rapid and robust data generation. An independent laser scatter channel permits labelfree detection and this can run concurrently with fluorescence measurements resulting in improved object recognition and fewer false negatives based on object count. This also enables beads of different sizes to be detected in addition to beads with different fluorescence encoding, resulting in unrivalled levels of multiplexing. The option to have dual laser excitation (488 and 640 nm) and four fluorescence data channels extends the range of commercially available dyes that can be combined in a single assay adding to the versatility of this system.

Mirrorball directly measures cellular or bead-based fluorescence on a well-to-well basis and also on an individual cell/bead basis. As such, Mirrorball lends itself to rapid screening for positive clones generated by hydridoma, SLAM and phage-display techniques. Only a small sample of cell supernatant is required (about 10μL), allowing multi-specificity profiling at the screening stage. Both whole antibodies and antibody fragments can be screened and the cellular assays can be performed on live or fixed cells from either adherent or suspension cultures. The automationfriendly benchtop design means it can be seamlessly integrated into existing workflows to ease screening bottlenecks. When combined with the abundance of compatible fluorescence reagents, Mirrorball offers a simple and easy solution to automated screening in laboratories developing antibody-based therapeutics.

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